Acute myelogenous leukemia (AML) results from a block in the terminal differentiation of normal myeloid precursors/stem cells leading to the potentially lethal accumulation of immature myeloblasts. Agents such as retinoic acid (RA) can enhance the terminal differentiation of certain myeloid leukemia cells and have had a major therapeutic impact in the treatment of the acute promyelocytic leukemia (APL) subset of myeloid leukemia. Nevertheless 20-30% of APL patients still relapse despite RA therapy, and most other types of AML fail to respond at all to RA. Defining factors which regulate RA receptor activity will provide insight into why these different AML cells respond differently to RA. We have observed that the Ca++regulated protein kinases (CaM kinases) are critical regulators of both RA receptor activity and myeloid leukemia cell differentiation. Indeed KN62, a small molecule inhibitor of the CaM kinases, triggers the in vitro terminal differentiation of certain AML cells. Our Long Term Objectives are to define the molecular basis for this previously unexplored role of the CaM kinases in regulating RA receptor activity and myeloid differentiation, and we hope to translate this knowledge into novel targeted therapy for certain human myeloid leukemias. In Specific Aim I we will determine the role of CaM kinase expression, cellular localization and enzymatic activity in regulating the differentiation of myeloid leukemia cells that are sensitive to RA and KN62 induced differentiation. In Specific Aim II we will explore CaM kinase expression, localization and activity in AML cell lines that are insensitive to RA/KN62 in an effort to biochemically and molecularly distinguish these insensitive AML cells from the sensitive cell lines analyzed in Specific Aim I. Finally in Specific Aim III we will assess the differentiative response of primary AML samples in short term culture to RA and KN62 and determine whether there is any correlation of their response with the specific clinicopathological or molecular parameters exhibited by these different primary AML cells. Relevance. These studies will dissect a new, previously unexplored role of the CaM kinases in regulating the differentiation of myeloid leukemia cells and are directly relevant for identifying new molecular targets for the clinical therapy of the human myeloid leukemias.